Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that impacts multiple diverse processes required during platelet activation. The overall goal of this project is to define the intracellular signaling pathways that Phosphatidylinositol Transfer Proteins (PITPs) contribute to the synthesis of phosphorylated phosphatidylinositols (phosphoinositides) in platelets, and to understand the role of PITPs in platelet biology. PITPs are a small protein family that has been found to bind and transfer phosphoinositide monomers from one cellular compartment to another in an energy-independent manner during vesicle trafficking and phospholipid signaling. Although there are no studies on the role of PITPs in hematopoietic cells, there is evidence in yeast cells that these proteins are essential for the biosynthesis and metabolism of phosphoinositides. Platelets have two dominant PITP family members, PITPa and PITPB. The overall hypothesis of this proposal is that the function of these PITP isoforms in platelets is not overlapping, and each is essential for the generation and spatial localization of discrete species of phosphoinositides within platelets. A secondary hypothesis is that the enzymatic activities of both PITP isoforms are necessary for normal platelet function ex vivo and in vivo. To understand the unique roles ofthe PITP isoforms in platelet biology. My laboratory has generated mice containing conditional null mutations in the PITPa and PITPP genes. The mature megakaryocytes and platelets of these mice lack PITPa, PiTPB, or both PITP isoforms, but the proteins are expressed normally in all other tissues. Loss of either isoform results in thrombocytopenia. Preliminary data also indicate that platelets lacking PITPa have a complete loss in the second messenger, lns(3,4,5)P3 (also known as 1P3) following stimulation by maximal doses of thrombin. In Aim 1 of this proposal, we will determine the link between PITP isoforms and phosphoinositide synthesis, as well as analyze the contribution of PITPs to platelet signaling. In Aim 2, i propose experiments designed to understand the distinct biochemical functions ofthe individual PITP isoforms. In Aim 3, we will study the role of PITP isoforms in platelet activation ex vivo and in vivo.